Truncated DNMT3B isoform DNMT3B7 suppresses growth, induces differentiation, and alters DNA methylation in human neuroblastoma

Abstract

Epigenetic changes in pediatric neuroblastoma may contribute to the aggressive pathophysiology of this disease, but little is known about the basis for such changes. In this study, we examined a role for the DNA methyltransferase DNMT3B, in particular, the truncated isoform DNMT3B7, which is generated frequently in cancer. To investigate if aberrant DNMT3B transcripts alter DNA methylation, gene expression, and phenotypic character in neuroblastoma, we measured DNMT3B expression in primary tumors. Higher levels of DNMT3B7 were detected in differentiated ganglioneuroblastomas compared to undifferentiated neuroblastomas, suggesting that expression of DNMT3B7 may induce a less aggressive clinical phenotype. To test this hypothesis, we investigated the effects of enforced DNMT3B7 expression in neuroblastoma cells, finding a significant inhibition of cell proliferation in vitro and angiogenesis and tumor growth in vivo. DNMT3B7-positive cells had higher levels of total genomic methylation and a dramatic decrease in expression of the FOS and JUN family members that comprise AP1 transcription factors. Consistent with an established antagonistic relationship between AP1 expression and retinoic acid receptor activity, increased differentiation was seen in the DNMT3B7-expressing neuroblastoma cells following treatment with all-trans retinoic acid (ATRA) compared to controls. Our results indicate that DNMT3B7 modifies the epigenome in neuroblastoma cells to induce changes in gene expression, inhibit tumor growth, and increase sensitivity to ATRA.

Figure 1. Aberrant DNMT3B expression and global DNA methylation in primary neuroblastoma tumors

A, RT-PCR examination of aberrant DNMT3B transcripts expressed in neuroblastoma (NB) and ganglioneuroblastoma (GNB) patient samples were normalized to the GAPDH housekeeping gene, and DNMT3B was amplified from exons 6 and 11 to identify the wild-type and aberrant DNTM3B transcripts specifically. DNA sizing is shown on the left and DNMT3B isoforms are shown on the right (ΔDNMT3B). B, RT-PCR examination of aberrant DNMT3B transcripts expressed in neuroblastoma cell lines, normalized to GAPDH housekeeping gene, and DNMT3B was amplified from exons 6 and 11 to specifically identify the wild-type and aberrant DNTM3B transcripts. DNA sizing is shown on the left, and DNMT3B isoforms are shown on the right (ΔDNMT3B). C, Identification of truncated DNMT3B proteins in S-type neuroblastoma cell lines by Western blotting. The positions of the molecular weight markers are given on the left. The positions of full-length DNMT3B and truncated DNMT3B7 are indicated on the right. GAPDH was used as a loading control.

Figure 2. Establishment of neuroblastoma cells overexpressed DNMT3B7

A, LA1-55n cells were transduced with Tet-controlled transactivator and subsequently with either a Tet-off inducible DNMT3B7 or control vector. One vector clone and two DNMT3B7 clones were grown in the absence of doxycycline for three weeks to induce expression. The cells were resuspended and counted every 3-4 days and plated at the same density. B, Whole cell extracts were made from the vector control and two DNMT3B7-expressing cell lines and subjected to Western blot analysis using an anti- DNMT3B antibody. Topoisomerase I was used as a loading control.

Figure 3. In vivo xenograft assays

A, DNMT3B7-expressing (Dox-) or control (Dox+) LA1-55n xenografts. B, H&E staining and immunohistochemistry of the LA1-55n xenograft sections. Control LA1-55n xenograft sections are on the left (Bi, Biii, and Bv) and DNMT3B7-expressing sections on the right (Bii, Biv, Bvi). H&E stained sections (Bi, Bii). CD-31 staining (Biii, Biv). Ki-67 staining (Bv, Bvi). C, DNMT3B7-expressing or control SMS-KCNR xenografts. D, H&E staining and immunohistochemistry of the LA1-55n xenograft sections. Vector control SMS-KCNR xenograft sections are on the left (Di, Diii, and Dv) and DNMT3B7-expressing sections on the right (Dii, Div, Dvi). H&E stained sections (Di, Dii). CD-31 staining (Diii, Div). Ki-67 staining (Dv, Dvi).

Figure 4. Global DNA Methylation by LC/MS

A, Global DNA methylation of a pure cell population of induced LA1-55n cells by LC/MS. B, Global DNA methylation of the LA1-55n induced xenograft tumors and SMC-KCNR constitutive xenograft tumors by LC/MS. C, Global DNA methylation of primary neuroblastoma tumors by LC/MS.

Figure 5. RNA-Sequencing Validation and CpG Island Methylation

A, Validation of over-expression of six genes in DNMT3B7-expressing cells by real-time RT-PCR. B, Validation of under-expression of six genes in DNMT3B7-expressing cells by real-time RT-PCR. C, Bisulfite sequencing of RXRB, a gene down-regulated in DNMT3B7-expressing cells. D, Bisulfite sequencing of EEA1, a gene up-regulated in DNMT3B7-expressing cells.

Figure 6. The effects of ATRA and DNMT3B7 expression on growth inhibition are additive

A, Growth of DNMT3B7-expressing or control cells treated with ATRA or vehicle. Expression was induced for two weeks and growth in ATRA or vehicle was determined for seven days. B, Validation of increased expression of GFRA1 and decreased expression of DLK1, both markers of differentiation, after treatment with ATRA or vehicle control, by real-time RT-PCR.